A submersion method to induce hypoxic damage in organotypic hippocampal cultures

Organotypic brain slices cultured on semi-porous membranes is an increasingly popular in vitro preparation for studying mechanisms of ischemic brain damage. To model in vivo hypoxia, cultured brain slices are exposed to anaerobic atmosphere by placing them into a special incubator. This requirement limits the use of in vitro ischemic models to highly specialized laboratories. Here, we describe a simple method that reproduces hypoxic injury, where cultured hippocampal slices are submerged into glucose-free deoxygenated medium for 1 h. The extent and distribution of hippocampal neuronal loss obtained with this treatment resembled that caused by hypoxia in living tissue in situ, i.e. CA1 pyramidal cell layer was most vulnerable and dentate granular cell layer was least susceptible to hypoxia as measured with fluorescence of the viability marker propidium iodide (PI). Electrophysiologic functional impairment determined by field recordings of CA1 pyramidal neurones temporally coincided with the extent of neuronal death. In addition, known neuroprotective treatments, such as hypothermia and phenytoin application ameliorated neuronal damage in a pattern similar to previously published reports. Therefore, the present in vitro model of ischemia is simple, reliable and of low cost. It is well suited for short and long-term studies of the mechanisms of hypoxic brain damage.